In mammals, calcineurin, the Ca2+/calmodulin dependent protein phosphatase, regulates immune cell activity, promotes heart and blood vessel development, mediates cardiac muscle response to stress, and modulates learning and memory in the brain. Calcineurin inhibitors, FK506 and cyclosporin A, are used clinically as immunosupressants, and, in animal models, reduce cardiac hypertrophy. As a key effector of Ca2+/calmodulin dependent signaling, detailed analysis of calcineurin function has the potential to impact many aspects of human health and development. In S. cerevisiae, calcineurin promotes survival during environmental stress, and in response to cell wall damage. A major role of calcineurin is to dephosphorylate and activate the Crz1p transcription factor, using mechanism analogous to calcineurin regulation of the mammalian transcription factor, NFAT. Additional calcineurin-mediated events that promote yeast survival during environmental stress are less well characterized, and are the focus of this application. This research aims to identify comprehensively the functions and components of calcineurin-dependent signaling pathways. We address these questions using S. cerevisiae, because of many experimental advantages offered by this simple eukaryotic organism, but strive to establish general principles that apply to calcineurin-dependent signaling in all cells. Previously, we exploited a particular feature of calcineurin signaling, i.e. the requirement for calcineurin to interact directly with its substrates via a docking site that is distinct from residues that are dephosphorylated, to identify several new components of calcineurin-mediated signaling pathways including 3 novel substrates: Slm1p, Slm2p and Hph1p. Here we propose to characterize further the mechanism by which calcineurin interacts with its substrates, which is evolutionary conserved. We will also apply genetic, genomic, and proteomic approaches to the identification of additional substrates and regulators of calcineurin. Specifically we will 1) Characterize calcineurin-docking sites in substrates and examine the impact of calcineurin-substrate affinity on signaling. 2) Examine the effect of specific point mutations in calcineurin on substrate interaction. 3) Identify the role of calcineurin interacting proteins in calcineurin signaling pathways. 4) Identify additional calcineurin substrates using novel proteomic and genomic screening methods.